Project Summary The co-PIs Loria and Batista from Yale will investigate the effect of temperature on allosteric pathways in the enzyme imidazole glycerol phosphate synthase (IGPS) from the hyperthermophilic bacterium T. maritima, at the molecular level, with emphasis on the influence of small molecule modulators that bind to the IGPS and affect the molecular mechanisms that synchronize the enzyme catalytic activity with effector N'-[(5'- phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binding at the allosteric site. IGPS is ideally suited for studies of allostery since it is a protein heterodimer, composed of the HisH and HisF proteins, with most of the properties of classical allosteric enzymes, including an oligomeric structure, multiple ligand binding sites, multiple conformational equilibria in the absence of ligand, and the stabilization of specific protein conformations by ligands. It is a potential therapeutic target since it is not found in mammals and is found in bacteria as well as in some plants and fungi. In particular many plant pathogens and opportunistic human pathogens such as Cryptococcus, Candida, and Ajellomyces that infect immunocompromised individuals have an IGPS that is highly homologous to the S. cerevisiae and T. maritima enzymes. Additionally, it has recently been shown that gene knockouts of HisF from Acinetobacter and Burkholderia pseudomallei increase the susceptibility of the former to ?-lactam antibiotics and lessen the infectivity of the latter. However, the role of entropy as reflected by unsual temperature effects on allosteric mechanisms that could represent targets for drug discovery has yet to be established. The research hypotheses are: (i) Higher temperatures and PRFAR binding increase flexibility in IGPS enabling conformational sampling of an active enzyme form; (ii) PRFAR-induced motions propagate through well- defined and conserved amino acid residues; (iii) Modulations of these motions and subsequent functional alteration can be achieved by small molecule allosteric ligands. The proposed methods combine Batista's computational modeling, including microsecond molecular dynamics simulations, network analysis, simulations of NMR spectra and computational drug screening, with Loria's state-of-the-art NMR relaxation techniques, quantifying the microsecond-to-millisecond conformational motions induced by drug or ligand binding with atomic resolution, mutagenesis studies, and isothermal titration calorimetry. The research program involves multiple cycles of an iterative approach where, in each cycle, allosteric pathways are explored through the analysis of differential motions probed by liquid-NMR relaxation methods and computation (MD and network analysis), obtaining valuable information on key amino acid residues and specific interactions responsible for transmitting structural or dynamical changes spanning the allosteric and active sites. The resulting insight provides guidelines for the next round of studies of mutants and modulators in a joint experimental and theoretical effort to elucidate the role of entropy on IGPS allosteric mechanisms.